Fighting bacterial pathogens with carbon nanotubes: focused review of recent progress

The fast and global spread of bacterial resistance to currently available antibiotics results in a great and urgent need for alternative antibacterial agents and therapeutic strategies. Recent studies on the application of nanomaterials as antimicrobial agents have demonstrated their potential for the management of infectious diseases. Among the diverse palette of nanomaterials currently used in biomedical applications, carbon nanotubes (CNTs) have gained massive interest given their many valuable properties, such as high thermal and electrical conductivity, tensile strength, flexibility convenient aspect ratio, and low fabrication costs. All these features are augmented by facile conjugation with functional groups. CNTs are currently available in many configurations, with two main categories being single-walled and multi-walled CNTs, depending on the number of rolled-up single-layer carbon atoms sheets making up the nanostructure. Both classes have been identified over the past years as promising antibacterial agents but the current level of understanding of their efficiency still harbors many pending questions. This mini-review surveys recent progress on the topic of antibacterial effects of CNTs and examines the proposed mechanisms of action(s) of different CNT typologies, placing the main focus on past studies addressing the antibacterial activity on Staphylococcus aureus and Escherichia coli, two prototypical Gram-positive and Gram-negative pathogens, respectively.


Introduction
Microorganisms are widely found in soil, water, plants, wild and domestic animals, humans, or foods. While most species are benecial for supporting current life on Earth, a small number are pathogenic and can cause disease to humans and animals. Many pathogenic microorganisms, especially those circulating in healthcare settings, have developed resistance to most of the available antibiotics and can cause severe infections accounting for tens of millions of deaths annually across the globe. 2,3 Among the most alarming pathogenic bacterial species, those comprised in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have gained the main focus of attention over the past decades. 4 These species are considered to be the most common bacterial pathogens in healthcare-associated (nosocomial) infections, causing extensive morbidity and mortality, especially in critically ill and immunocompromised patients. 5,6 ESKAPE pathogens are characterized by a high level of antibiotic resistance, 7 which recently prompted the World Health Organization to list them among the greatest threats to human health and to encourage research on new effective drugs for the treatment of antibiotic-resistant infections, 8 which are urgently needed.
The great genetic adaptability, intrinsic bacterial resistance genes, and the selective pressure exerted by the massive use of antibiotics are responsible for the appearance, transfer, and spread of antibiotic resistance genes and bearing strains. 9 Other factors contributing to the emergence of drug-resistant strains are thoroughly discussed in the recent review of Larsson et al. 10 Recently, nanomaterials emerged as important tools in the ght against multidrug-resistant (MDR) bacteria. 11 These materials can be used as "nano-weapons" that can act individually or in synergism with antimicrobial compounds against bacteria. This synergism holds valuable intrinsic potential for the development of next-generation, all-in-one agents, that can combat both drug-susceptible and MDR strains. The most common mechanism of action of nanomaterials relies on their interaction with the cellular envelope of bacteria, causing its destabilization and ultimately cell death, even for highly resistant species. 12 Currently, among the most studied nanomaterials proposed as alternative antibacterial agents, metal-based nanoparticles (NPs), graphene-based nanomaterials, and carbon dots have extensively been demonstrated to have signicant antibacterial properties (more comprehensively reviewed by Dong et al., 13 Sánchez-López et al. 14 and Zhang et al. 15 ).
Given their size and selectivity for bacteria, metal-based nanoparticles (NPs) have proved to be highly effective against the pathogens 14 listed as a priority by the World Health Organization. Among them, silver-based NPs represent maybe the most effective antibacterial agents in this class, while NPs carrying other metals (i.e., gold, zinc, copper, etc.) have been observed to exert variable bactericidal activities. 14 Graphene-based nanomaterials have been developed for many purposes spanning from the promotion of bacteria proliferation to microbial inhibition. These materials have been used as growth promoting agents of bacteria to accelerate interspecies electron transfer during anaerobic metabolism. On the other hand, graphene-based materials with antibacterial properties have been synthesized to prevent biolm formation on membranes for water treatment, medical equipment, and tissue engineering scaffolds. 15 Carbon dots, constituted by small carbon nanoparticle cores with adsorbed surface passivation molecules, are generally nontoxic. However, with their effective light-harvesting properties over a very broad spectral range from UV to near-IR, carbon dots have exhibited strong photodynamic antibacterial effects. 13 Next to these nanomaterials, carbon nanotubes (CNTs) have also been demonstrated as highly efficient antibacterial agents over the past years. CNTs are cylinder-shaped allotropic forms of carbon, with diameters of several nanometres and lengths ranging from nanometres to tens of centimetres, [16][17][18][19] depending on the targeted application and the employed synthesis protocols. CNTs originate from graphene sheets, whose layers appear as a rolled-up, continuous, hexagonal-like mesh structure, with the carbon molecules positioned at the apexes of the hexagonal structures ( Fig. 1). CNTs with walls comprised of a single graphene sheet are known as single-walled carbon nanotubes (SWCNT) (Fig. 1A), while multi-walled carbon nanotubes (MWCNT) originate from the rolling up of several graphene layers 20 (Fig. 1B). SWCNTs-and MWCNTs-based materials can be obtained by different preparation methods relying on chemical vapor deposition, laser ablation, ame synthesis, NPassisted catalytic synthesis, and others, [20][21][22] which results in important advantages, such as low-cost and wide-availability. A rich palette of protocols for functionalizing both SWCNTs and MWCNTs has been described in the literature to date, 23 and among the various applications of obtained CNTs, their utility as therapeutic agents against MDR bacterial infections is generally acknowledged, holding great promise in the quest for next-generation antibacterial strategies that can lower antibiotic dosage or, in some cases, entirely replace the use of drugs. 24 Importantly, in the context of ghting bacteria, CNTs have not been used only as antimicrobial agents but have also demonstrated important usefulness in sensing applications. 25,26 Due to their great adaptability 10 and excessive use of antibiotics 27 over the years, microorganisms have developed different MDR phenotypes. In this focused review we discuss past efforts that were aimed at demonstrating the use of SWCNTs and MWCNTs as antimicrobial agents against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), which have proled over the past years as two prototypical Gram-positive and Gramnegative models. S. aureus is a Gram-positive bacterium, which causes a variety of mild to extremely severe infections, that is widely acknowledged as an important source for the spread of many antibiotic resistance genes worldwide. 28 E. coli is a species that is responsible for diverse pathological conditions with health hazards ranging from mild to severe, 29,30 with drugresistant E. coli strains posing a signicant global threat. 31,32 While antibiotic drugs remain the gold-standard in the ght against bacterial infections, the use of various nanomaterials as alternative solutions has been thoroughly explored in past studies. 33,34 Most of these studies concluded that Gram-negative species are more resistant to membrane damages caused by nanomaterials than Gram-positive bacteria, given additional protection provided by their outer membrane. 35 CNTs were as well considered in these past efforts, having been shown to be highly capable of severly damaging the cellular envelope (cell wall and membrane) leading to leakage of cytoplasmatic content and consequent cell death. 36 While important work that has signicantly contributed to the current level of understanding of the interactions taking place between CNTs and bacteria is also presented as background in the next section, we place the main emphasis on discussing selected works published in the past ve years, that illustrate various facets on the use of SWCNTs and MWCNTs in antimicrobial applications. Considering that the use of these materials in their pristine form is known to present a series of limitations linked to aspects such as chemical inertness, hydrophobic character, poor adhesion during interaction with the cell wall, and instability in aqueous solution, we also focus our attention on SWCNTs and MWCTNs whose antibacterial properties were enhanced by the addition of functional groups via covalent and non-covalent bonds. In many cases, such strategies were found useful to reduce the dose of CNTs, and to achieve higher therapeutic efficacy compared to antibacterial solutions building on pristine CNTs. Overall, we consider this review to be a useful resource for those interested to get acquainted with the topic of antibacterial CNTs, and for peers interested in a glimpse on the current state-of-the-art.

Pristine SWCNTs as antimicrobial agents
Despite convincing evidence reported to date on the antibacterial role of SWCNTs, a dened mechanism of action has not been univocally demonstrated so far. 24 Many studies have attributed the antibacterial properties of SWCNTs to a wide range of potential mechanisms including metabolic alteration or inhibition, 37 oxidative stress, 38 and physical piercing damage to the cell envelope. 39 Work performed to date has also shown that the antibacterial activity of CNTs can be inuenced by: (i) structural properties (such as diameter, length, aggregation, and surface functional groups), (ii) concentration; (iii) buffer solution in which SWCNTs are solved; (iv) exposure time to SWCNTs, or (v) the number of collisions and extent of the impact forces occurring between the SWCNTs and the bacterial cells. 36 With respect to the latter, in a landmark study reported by Kang et al., 1 it has been proposed that cell envelope damage, resulting from direct physical contact between bacteria and SWCNTs, represents in fact the major cause of bacterial death. The authors showed that SWCNTs exhibit stronger antibacterial activity than MWCNTs against E. coli K12, speculating that this probably relates to their smaller diameter size that facilitates partitioning and partial penetration into the cell envelope. However, the relationship between SWCNTs length and antimicrobial effects remains controversial: Aslan et al. 40 demonstrated that the use of shorter SWCNTs caused additional damage to the surfaces of E. coli cells, which they hypothesized to be most likely related to the increased chances for interaction between the SWCNTs open ends and the targeted microorganisms. On the other hand, Yang and co-workers 41 found that longer SWCNTs have stronger antimicrobial activity against the pathogenic bacterium Salmonella typhimurium due to their improved aggregation capability with bacterial cells, in a study that discussed as well limitations of short SWCNTs to bind to bacterial cells. With respect to other physical properties, Chen et al., 42 addressing in their work a broad-spectrum of CNTs, proposed that rigid and thin SWCNTs are more effective in terms of cell wall and membrane piercing of round-shaped bacteria than MWCNTs. An open debate still exists on what is the optimal CNT conguration for achieving the most efficient antibacterial effect.
Next, we discuss several relevant works, placing main focus on those published over the past ve years, in which the antibacterial effects of SWCNTs, either in pristine form, or conjugated with other nanomaterials, or antibiotics, were discussed with respect to their activity on S. aureus and E. coli strains.
Among the wide palette of endeavours reporting SWCNTbased antibacterial tools, the work of Basiuk et al. 43 investigated the antibacterial effects against S. aureus of pristine SWCNTs in comparison with nanodiamond graphene (ND) and graphene oxide (GO), two alternative carbonaceous nanomaterials that have gained increasing attention due to their presumed better biocompatibility compared to CNTs in specic scenarios, discussed in previous works. 44 The authors found that among the tested nanomaterials, pristine GO exhibited the most pronounced antibacterial effects, exhibiting a dosedependent behaviour. SWCNTs showed activity against S. aureus, but only at high concentrations (1 and 10 mg mL −1 ), while pristine ND was found not only to be less toxic but also to promote bacterial growth at the highest concentration assayed (10 mg mL −1 ). We consider this study to be important as it shows that carbonaceous nanomaterials exhibit consistently different antibacterial effects depending on their size and geometric conguration.
Noor et al. 39 have addressed in their study the fact that SWCNTs are usually difficult to disperse, and thus many studies focusing on their antibacterial effects use them in combination with dispersion aides. While many of these contribute themselves to the exerted bacterial stress, many times such aspects are unaccounted for. The authors discussed thus the antibacterial effects of SWCNTs when administered with ve dispersant agents: sodium dodecyl sulfate (SDS), Pluronic, lysozyme, DNA, and tryptic soy broth (TSB). They observed that SDS is fatal to S. aureus regardless of the presence of SWCNTs, while the activity of Pluronic and lysozyme against S. aureus was enhanced by the presence of SWCNTs. In contrast, DNA and TSB dispersions did not have any activity regardless of the presence of SWCNTs. Overall, the work of Noor et al. 39 showed that studies focused on assessing the antibacterial activity of SWCNTs need to carefully consider the synergistic interactions taking place between these nanomaterials and dispersants, which may result in different levels of stress exerted on cells compared to the case when pristine SWCNTs are used without dispersion agents.

Composite and functionalized SWCNTs as antimicrobial agents
The promising effects of pristine SWCNTs against bacterial pathogens prompted to the generation of functionalized forms of these CNTs to optimize their activity. In this body of efforts, Sah et al. 45 have introduced as an efficient photodynamic antimicrobial chemotherapeutic agent a nano-composite made up of SWCNTs and amine-functionalized porphyrin. They showed that upon exposure to visible light, the porphyrin conjugated nanotubes inict damage to S. aureus bacterial cells, nally leading to their death. Field emission scanning electron microscopy (FESEM) images of the cells treated with the photoactivatable nanocomposite showed the formation of web-like structures on the affected cells, which were dependent on the light irradiation time. This study showcases that the intrinsic antibacterial effects of SWCNTs can be signicantly augmented by conjugating them with photosensitisers. In a different study addressing the use of SWCNTs in combination with photoactive nanomaterials, Mohammad et al. 46 coated pristine SWCNTs with Ag-doped TiO 2 NPs with a size estimated to range between 7.7 and 13.53 nm, which were found to be responsible for antibacterial photocatalytic effects. These nanocomposites were tested against bacterial strains of both E. coli and S. aureus models, with the authors observing Gram-negative bacteria to be more resistant to the proposed nanocomposite compared to Gram-positive bacteria, under illumination by UV light. MWCNTs were evaluated as well in this study, providing less efficient results. The authors speculate that although MWCNTs-TiO 2 /Ag showed a slightly lesser toxicity against bacteria in their experiments, in specic applications, they might represent the better choice given their more reducedfabrication costs. Additional insights on CNTs based antibacterial phototherapies can be found in the thorough review work of Wang et al. 47 Considering other antibacterial routes, Zhu et al. 48 introduced an ingenious antibacterial nanoplatform consisting of SWCNTs decorated with silver nanoparticles and coated with mesoporous silica. They showed that the outer mesoporous silica shells improve the dispersibility of SWCNTs, increasing their contact area with bacteria cell envelope, while the large number of mesopores in the silica layers act as microreactors for in situ synthesis of Ag NPs with controlled small size and uniform distribution. They compared the effects of this nanocomposite with the antibacterial properties of mesoporous silica coated SWCNTs and commercial Ag NPs, observing much stronger antibacterial performance against MDR S. aureus and E. coli strains, due to the larger extent of damage to the bacterial cell membranes, Fig. 2A, and the faster release of silver ions. Importantly, they also tested this nanocomposite in vivo using a rat skin wound infection model, showing remarkable bacterial clearance capabilities for MDR S. aureus strains, accompanied not only by great biocompatibility but also by valuable wound healing effects, Fig. 2B, which are known to be correlated with bacterial load. 49 Besides past studies reporting the combination of SWCNTs with other nanomaterials to obtain more efficient therapeutic tools against bacteria, it is important to note that SWCNTs have also been successfully conjugated for this purpose with antibiotics. For example, Carver et al. 50 proposed SWCNTs and nanographene oxide (NGO) as solutions for delivering the antibiotic tetracycline to a tetracycline-resistant E. coli strain. Tetracycline loaded-SWCNTs and NGOs were found to inhibit this strain, even for tetracycline amounts much lower compared to the minimum inhibitory concentration (MIC) of free tetracycline. This was attributed to the capacity of these two carbonaceous nanomaterials to transport the antibiotic into the cells and thus to circumvent the drug-resistance mechanism based on the expression of efflux pumps. SWCNTs were more efficient in delivering tetracycline compared to NGOs, which was attributed to their needle-like shape. This study consolidates the current belief that nanomaterials may represent a cornerstone for nextgeneration antibacterial therapies, showing that besides their intrinsic antibacterial properties, they can augment the effects of antibiotics, enabling their administration in lower doses, helping to reduce the selective pressure, and overcome antibiotic resistance.
In a study reported by Sapkota et al. 51 the authors exploited for antibacterial purposes the fact that SWCNTs can be easily chemically combined with various semiconductor nanostructures such as ZnO, ZnS, SnO 2 , CdS or CuO. Considering the latter, they fabricated SWCNT-CuO nanocomposites by straightforward recrystallization accompanied by calcination, which resulted in heterojunctions being formed between the SWCNT surface and the CuO nanocrystals that were chemically attached to the SWCNT surface. Antimicrobial susceptibility assays demonstrated excellent bactericidal properties of the proposed material on both E. coli and S. aureus models. The authors attributed the bactericidal effects to the increased intracellular concentration of reactive oxygen species (ROS) resulted from the occurring chemical reactions, which are known to kill bacteria through cumulative oxidative stress. 52,53 However, it is possible that endogenous ROS, produced by bacteria in response to membrane damage by SWCNT-CuO nanocomposites, may contribute to bacterial killing. 54 Another study explicitly nominating ROS as the main antibacterial mechanism, is the work of Rugaie et al., 55 where the authors laced ZnO-Ag and ZnO-Au nanocomposites into SWCNTs to yield ZnO-Ag-SWCNTs, and ZnO-Au-SWCNTs. They showed that pre-treatment of phagocytic cells with these nano-hybrids activates these cells, enhancing phagocytosis and microbicidal activity by ROS and NADPH oxidase production. Moreover, this study demonstrated that ZnO-Ag-SWCNTs and ZnO-Au-SWCNTs nanocomposites contributed to the bactericidal activity against E. coli to a greater extent than the SWCNTs alone, Fig. 2C, as shown by the enhanced, excessive production of ROS, which is considered to be derived from increased NOX2 activation. This study thus highlights that SWCNT-based nanocomposites can stimulate the antibacterial response by the host innate immune system.
In a study addressing a different antibacterial function of SWCNTs, Kumar et al. 56 functionalized pristine SWCNTs through acidic treatment for nucleation, followed by reduction of silver ions by microwave heating to produce Ag-NPs decorated SWCNTs (Ag-SWCNTs). Via a dip-dry-curing process, they coated on different cotton fabrics pristine SWCNTs, Ag-NPs and Ag-SWCNTs composites and qualitatively evaluated the antibacterial property of all coated fabrics against S. aureus and E. coli. The Ag-SWCNTs coated fabrics showed excellent antibacterial activity against both types of bacteria (the highest in the tested group), which did not signicantly diminish even aer many washings. This study represents an important example on the usefulness of SWCNTs-based nanocomposites to enable a next generation of fabrics that can prevent contamination/ infection with bacteria, which can be especially useful in bacteria rich environments, such as hospitals. Table 1 summarizes the main features of the described pristine and composite SWCNTs.

Fighting bacterial pathogens with MWCNTs: focused overview
Pristine MWCNTs as antimicrobial agents MWCNTs are hollow cylindrical carbonaceous nanomaterials with walls composed of more than one sheet of graphene, with a typical diameter ranging in the order of tens of nanometres (Fig. 1). Among other signicant roles that the nanotechnology community has identied for them, MWCNTs are regarded as promising tools in nanomedicine, 57 with manyfold uses including as delivery vehicles for metallic nanoparticles, drugs, DNA aptamers, peptides, and proteins. 58,59 Their intrinsic antibacterial properties are also widely acknowledged. 24 Although it has been speculated that many of their cytotoxic effects in bacteria depend on multiple mechanisms, correlated with diverse factors such as amorphous carbon content, catalytic metal content, bundled conformation, length, and dispersity in aqueous media, 60 the exact mechanisms behind their antibacterial modes of action are yet to be understood, mainly due to the scale at which MWCTNs-bacteria interactions take place, with many aspects unavailable to the resolution limitations of currently available characterization techniques. Similar to the mechanisms of actions acknowledged for SWCNTs, MWCNTs have been proposed to have membranolytic activities and/or to induce the production of ROS, which results in bacterial death due to massive oxidative stress. 52,53,61 However, various studies proposed additional mechanisms over those of SWCNTs. For example, Mocan et al. 62 suggested that the release of impurities upon MWCNT exfoliation may hold an important role in bacterial killing.
In a study reported by Saleemi et al., 63 it was shown that double-walled CNTs (DWCNTs) and MWCNTs inhibit the growth of many different opportunistic pathogens, including S. aureus, P. aeruginosa, K. pneumoniae, and fungal strains belonging to Candida albicans. Importantly, it was shown that the evaluated CNTs selectively damage the microbial cell walls or membranes, Fig. 3A, depending not only on the conguration of the nanotubes but also on the pathogen morphology. While studies focused on SWCNTs 40 suggested that shorter CNTs are likely to induce more damage to bacteria, due to a higher chance of rupturing the envelope by the sharp ends, here it was hypothesized and partially demonstrated that longer CNTs may be more efficient as they wrap around the surface of the pathogen cell, yielding a higher surface contact area with the cell wall compared to shorter CNTs, with a proportional increase of efficacy. The authors also evaluated DWCNTs and  Among the evaluated MWCNT instances, the highest antimicrobial activity (in terms of the largest diameter of inhibition zone) was observed for MWCNTs decorated with ZnO and Ag NPs. The biolm formation assay also demonstrated that these two variants exhibit inhibition of biolm formation, consolidating the idea that antimicrobial systems building on MWCNTs and Ag and ZnO NPs are valuable solutions to be considered in the ght against resistant pathogens and biolm associated infections. In another study discussing the effects of MWCNTs on biolms, Abo-Neima et al., 66 showed that MWCNTs functionalized via an interaction with nitric acid were able to prevent E. coli and S. aureus biolm formation. Furthermore, these materials were found to be capable to disrupt mature biolms leading to their detachment. Transmission electron microscopy images revealed morphological changes that reect the damage mechanisms. The functionalized MWCNTs were found to biologically isolate the cells from their surrounding microenvironment, contributing to the development of toxic substances and placing the cells under oxidative stress, nally leading to their death. The antimicrobial and biolm formation resistance properties of MWCNTs were also demonstrated in the study of Madenli 67 et al., who studied MWCNTs blended polyethersulfone (PES) membranes, considering as model of target organisms E. coli and P. aeruginosa. Their results showed that, following the deposition of E. coli cells onto the membrane surface, no colonies were formed on composite membranes instances synthesized at particular MWCNT content levels, whereas for membranes of similar composition incubated in P. aeruginosa suspensions, consistently less biolm formation occurred within 24 h. Importantly, the authors showed no MWCNT release during the water ltration of the composite membranes, which is important in light of potential applications for separation and purication.
As discussed also in the previous section, addressing SWCNTs, CNTs have a high potential to enable efficient antibacterial photodynamic therapies. 47 In this context, Parasuraman et al. 68 assessed an antimicrobial photodynamic therapy based on methylene blue-conjugated MWCNTs (MBCNTs) on biolms of E. coli and S. aureus, Fig. 3B. Under illumination with a laser source emitting at 670 nm, biolm inhibition, cell viability, and extracellular polymeric substances (EPS) reduction assays showed higher inhibition in S. aureus than in E. coli. This was found to be correlated with the fact that the binding and uptake of MBCNTs was greater in S. aureus compared to E. coli, which was consistent with previous work addressing the killing of Gram-positive and Gram-negative bacteria with nanoplatforms incorporating methylene blue. 69 Another study evaluating an antibacterial photodynamic therapy based on MWCNTs has been performed by Cuadrado et al. 70 They studied two magnetic nanocomposites based on GO and MWCNTs loaded with the photosensitiser menthol-zinc phthalocyanine (ZnMintPc). These were conjugated with iron magnetic nanoparticles and encapsulated in a lipophilic envelope, conferred by treatment with a biocompatible hydrogel based on N-vinylcaprolactam (VCL) and poly(ethylene glycol)diacrylate (PEGDA), used to help with the dispersion of the considered hydrophobic compounds in aqueous media. The two magnetic nanocomposites, VCL/PEGDA-MNPs-MWCNTs-ZnMintPc and VCL/PEGDA-MNPs-GO-ZnMintPc, were found to exhibit excellent photodynamic/photothermal effects under 630 nm illumination against E. coli, S. aureus (Fig. 3C), and C. albicans. While VCL/PEGDA-MNPs-GO-ZnMintPc nanocomposites were efficient only against E. coli and S. aureus the VCL/PEGDA-MNPs-MWCNTs-ZnMintPc instances were able to suppress all these three pathogens, demonstrating their broad-spectrum as antimicrobial agents building on photodynamic and photothermal effects. Considering recent progress reported on the topic of cancer cell killing via magneto-mechanical forces exerted by endocytosed magnetic nanoparticles, 71 we have reason to believe that such strategies may soon become reality also in the context of antimicrobial applications. This may represent an important breakthrough given the complementarity of magneto-mechanical and photodynamic/photothermal therapies. 72 In another relevant effort, Baek 73 et al. exploited the fact that metal oxides are known to increase mobility, surface area, and photocatalysis when combined with CNTs. Specically, they evaluated the antibacterial effects of ZnO-and TiO 2 -conjugated MWCNTs and GO nanocomposites in relationship to E. coli. The ZnO-based nanocomposites exhibited a higher antibacterial role compared with the TiO 2 based instances, with the authors obtaining antibacterial effects in terms of bacterial cell growth inhibition in the order ZnO-GO > ZnO-CNT > TiO 2 -GO > TiO 2 -CNT. This study also focused on identifying which of the four possible antibacterial mechanisms is mainly responsible for the observed antibacterial effects: (i) generation of ROS, (ii) physicochemical characteristics, (iii) the steric effect, and (iv) release of metal ions. ROS generation was found to be in lead over the others, with the physicochemical characteristics and the steric effect taking part of the contributing mechanisms as well. This study suggests also that GO-based nanocomposites are to be preferred over CNT-based nanocomposites, with Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) images revealing that GO-based nanocomposites exhibited better attachment to the bacterial surface, while CNT-based nanocomposites signicantly aggregated to each other, diminishing thus the interaction chances with the cells. Another relevant study focused on ZnO-MWCNT nanocomposites has been reported by Shakir et al., 74 who evaluated Co doped-ZnO/MWCNTs nanocomposites synthesized by means of the sol-gel method. They evaluated various modications occurring in the physical properties of instances synthesized under different Co doping concentrations. They observed the growth of spherical clusters over the surface of interlocking cylindrical tubes, and that the Co doped-ZnO/ MWCNT hybrid nanocomposites exhibit high absorbance, and band gap narrowing upon increasing cobalt-doping concentration, which can facilitate a wide range of applications. With respect to the antibacterial effects, the authors observed high inhibition efficiency for instances synthesized under high concentrations of Co, for both S. aureus and E. coli models. Given that cobalt is known for good biocompatibility and low toxicity, the nanomaterials discussed in this study represent an interesting example on the synergy between CNTs and Co.
Same as SWCNTs, MWCNTs can also be used in combination with conventional antibiotic drugs. For example, Hassani et al. 75 introduced a novel nano-drug synthesized by covalent graing of modied MWCNTs with levooxacin (LVX). The MWCNT-LVX agent was demonstrated to be highly efficient against S. aureus strains. The novel synthetic nano-drug possessed high loading capacity and pH-sensitive release prole in vitro and in vivo, exhibiting higher bactericidal activity in a mouse S. aureus burn wound infection model compared to the stand-alone use of LVX. 75 In a different type of approach, Bains et al., 76 exploited the fact that ionic liquid (IL) and MWCNTs show signicant synergistic effects given the occurring strong p-cation interactions. 77 They developed a material based on IL-functionalized MWCNTs for hydrophobic coatings, showing their effectiveness over S. aureus (including a methicillin-resistant strain), and E. coli. By the help of atomic force microscopy (AFM) and SEM, they elucidated the mechanisms of action, Fig. 3D, which conrmed the motivation of their design which was selected given the hypothesized electrostatic interactions through the cationic moiety with the negatively charged bacterial membrane, and the cell enveloped damage potentially favoured by the considered hydrophobic carbon chain length. The proposed material was also evaluated as a coating on a PVC  substrate, a scenario in which it exhibited remarkable inhibition of the bacterial cell growth in vitro. Overall, this study has great potential to favour the advent of next-generation antimicrobial surfaces with self-sterilizing abilities. Other important applications of CNTs in the context of antimicrobial surfaces are nicely presented in the recent review of Teixeira-Santos et al. 78 Moskvitina et al. 79 assayed different carbon-based nanomaterials and demonstrated that carboxyl-functionalized MWCNTs are endowed with a strong antibacterial potential against E. coli and S. aureus in terms of growth inhibition and alteration of membrane integrity, presenting an activity comparable to catalytic lamentous carbon with different orientations of graphene blocks, ionic carbon, and ultrane explosive NDs.
Finally, we nd noteworthy to highlight the mechanobactericidal action of vertically aligned MWCNTs (VAMWCNTs), which was demonstrated in the landmark work of Linklater et al. 80 In their study, the authors showed that VAMWCNTs arrays inactivate both Gram-negative (P. aeruginosa) and Gram-positive (S. aureus) bacterial cells. The mechanistic action leading to the bacterial cell death stems from the elasticity of the proposed nanostructures, exhibiting a high aspect ratio (100-3000) between their length (microns) and diameter (approximately 10 nm). The authors demonstrated that upon the adsorption of bacteria onto the nanostructured surface, the deection and retraction of MWCNTs results in physical membrane perturbation and cell death (Fig. 3E). In the context of the current efforts devoted to developing antibacterial surfaces building on mechano-bactericidal effects, 81,82 we argue that CNTs are likely to play an important role in the years to come for enabling such applications. Table 2 summarizes the main features of the described pristine and composite MWCNTs.

Brief considerations on current challenges in fighting antibiotic resistant strains with CNTs
In Fig. 4 we display the main putative antibacterial mechanisms of action of CNTs, deduced from all the results discussed in this work. However, the absence of an unambiguous model raises the need for further light to be shed on the exact mechanisms caused at the molecular level by the upon bacteria interaction with CNTs. As the antibacterial mechanisms of CNTs seem to involve multiple cellular targets 23,24 (i.e. cell wall, cell membrane, modications of proteins and DNA by ROS-induced damages, etc.), it seems improbable that bacteria strains could develop resistance to these nanomaterials. However, to what extent a selection of resistant mutants could withstand the action of CNTs remains a puzzling question, which denitely warrants the requirement of further studies on the subject. However, it is important to note that bacteria could potentially develop signicant morphological changes, which could impact the antibacterial efficiency of CNTs. Indeed, it seems that there is a strong relation between antibiotic resistance and MWCNT effect on S. aureus cells. The antimicrobial efficiency of MWCNTs seems to be lower in MDR strains, as compared to antibiotic-susceptible isolates. S. aureus strains resistant to antibiotics acting on cell wall are less susceptible to both pristine and functionalized MWCNTs. 83 The idea that cell wall structural modications could interfere with the antimicrobial activity of CNTs is also supported by the work of Hassani et al., 75 discussed in the previous section, which reports different antibacterial efficiency of the MWCNTs depending on the cell wall structure of the target cells.
The antibacterial activity of nanomaterials in highly organized multicellular communities is also a common challenge, as bacteria in biolms behave completely different compared to their planktonic counterparts. Microbial biolms are more tolerant to all known antimicrobials and host defence mechanisms; therefore, the management of biolm-associated infections is challenging. 84 A recent study 85 showed that MWCNTs promote bacterial conjugative plasmid transfer in aqueous environment. The results of this study suggest that the presence of particular MWCNTs congurations, especially clustered, provide bacteria with novel surfaces for intense cell-to-cell interactions in biolms and can promote bacterial horizontal gene transfer, hence potentially elevating the spread of antimicrobial resistance. This leads to the idea that results obtained in studies addressing planktonic bacteria cannot be straightforward extrapolated to applications addressing biolms, therefore knowledge transfer between these two elds of research should be done with extreme caution.

Conclusions
Due to the increasing number of drug-resistant bacterial strains and their high pressure on the sustainability of health system across the globe, there is an urgent need to reduce the exposure to antibiotics, which is known to favour the development of resistance mechanisms. To this end, nanomaterials have been widely explored to date as solution to replace antibiotics, or as means to enable lower antibiotic doses. In the frame of these efforts, CNTs have been found to hold important antibacterial potential. Knowledge on the mechanisms by which CNTs are capable to kill bacteria is constantly growing, together with the extent of functionalization routes that results in enhanced antimicrobial effects. In this focused review we have discussed recent progress on the use of SWCNTs and MWCNTs as the backbone of various antibacterial solutions and tools, placing main emphasis on works reported over the past ve years. The discussed works showcase various ways by which SWCNTs and MWCNTs can contribute to overcoming the current crisis that humanity faces due to the emerging number of drug-resistant microorganisms. We hope that our work will inspire future research aimed at understanding in more detail the interactions taking place between CNTs and prokaryotic cells, and the advent of novel CNT-based "weapons" capable to efficiently ght drug-resistant and drug-susceptible bacteria.

Author contributions
All authors identied relevant references and designed the structure of the manuscript. MA, ML, CC, AMH, PV and SGS wrote the initial dra. SGS, MA, ML and PV have prepared the gure artwork. ML and PV have prepared the tables. All authors read and revised the scientic content of the initial dra, and of the revised version, and helped on reorganizing text to promote clarity. SGS and PV coordinated the overall effort.

Conflicts of interest
There are no conicts to declare.